Biochemistry and Molecular Biology of Blood Coagulation
Our laboratory has focused on understanding the metal- and membrane binding properties of the vitamin K-dependent proteins. This laboratory has made substantial progress in the determination of the structures that define vitamin K-dependent protein-membrane interaction. We recently solved the structure of the Gla domain of human Factor IX by 2D NMR spectroscopy, obtaining a high resolution structure in the presence of calcium and in the presence of magnesium. By comparing these structures, we concluded that residues 1 to 11 include a contact site for phospholipid binding since the calcium-bound conformer interacts with phospholipid whereas the magnesium-bound conformer does not. Using a photoaffinity label, the close (< 3) proximity of leucine 6 and phenylalanine 9 in Factor IX to the phospholipid bilayer was demonstrated. In collaboration with Dr. Barbara Seaton, we have recently crystallized bovine prothrombin fragment 1 in the presence of phospholipid analogs and calcium ions. A model of phospholipid-prothrombin interaction has been developed that incorporates a phosphoserine binding site and residues 1 to 11, including the hydrophobic patch composed of three hydrophobic residues (phenylalanine 4, leucine 5 and valine 8) and the acyl chains from phosphatidylserine. This "phosphoserine mooring" model is consistent with known information about the interaction of the vitamin K-dependent proteins and phospholipid membranes, including the requirement for anionic phospholipids, the hydrophobic patch, calcium ions and -carboxyglutamic acid. Refinement of this model by Xray crystallography and NMR spectroscopy coupled to biochemical studies of protein-membrane interaction remain major areas of interest to understand protein complex formation on membrane surfaces during blood coagulation.